Of the objectives identified for the entire duration of the project, those to be pursued in the third year are: (1) Description of the enzymatic specificities of the proteases isolated from the 3 canine and 6 rainbow trout zymogens that we have already prepared. These studies will compare the activities against small peptide substrates that have been useful in distinguishing the acid proteases secreted as zymogens by porcine and bovine stomachs (pepsin, chymosin, gastricsin). (2) Analyze the mechanism of activation of porcine pepsinogen by using a spin-labeled zymogen and by stopped-flow kinetics. This study will detect ESR and UV spectral changes, development of enzymatic activity, and cleavage of peptide bonds of the zymogen during activation. (3) Prepare antibodies to the 3 canine zymogens in anticipation of studies on secretion of these zymogens and their distribution throughout the gastrointestinal tract. (4) Continue chemical studies on the composition and amino acid sequences of the canine and trout zymogens. The carbohydrate content of the proteins will be more fully characterized.